The long term objective is to understand the mechanism of Ca2+ transport by the Ca2+ ATPase of sarcoplasmic reticulum. Expression and mutagenesis of full length or partial cDNAs encoding the Ca2+ ATPase, followed by assay of the function of the product, is being used to address questions of structure/function relationships in the protein. Research is proposed under six headings: 1. Determination of the roles of transmembrane amino acids in Ca2+ binding. The mutation, E309Q, abolishes one of two Ca2+ binding sites and this defective site is 'stacked' on the cytoplasmic surface of the membrane. The demonstration that other Ca2+ binding and Ca2+ affinity mutations also abolish one of two Ca2+ binding sites and are 'stacked' on either cytoplasmic or lumenal surfaces is proposed. 2. Analysis of Ca2+ binding 'patches' in transmembrane sequences. Analysis of functional consequences of mutations throughout all of M4 has revealed an active Ca2+ binding patch. The demonstration of similar active 'patches" in each of M5, M6 and M8 and use of the information to refine models for the Ca2+ binding sites is proposed. 3. Determination of the topology of Mg/M10. The hypothesis that Mg and M10 are transmembrane sequences will be tested by investigation of their location and function. 4. Expression, purification and crystallization of cytoplasmic domains. The cytoplasmic sequence between N330 and D738, which can expressed stably in E. coli and which binds TNP-ATP, will be purified in sufficient quantity to initiate attempts at crystallization for high resolution X- ray diffraction analysis of the structures of domains contained in the sequence. 5. Identification of ATP binding residues by measurement of TNP-ATP binding. TNP-ATP binding to wild-type and mutant ATPases, will be developed as a more definitive test for the involvement of specific residues in ATP binding. The hypothesis that the B-strand domain plays a functional role in energy transduction will be tested using mutants and chimeras. 6. Determination of a potential regulatory role for acidic residues in stalk sector 2. Chimera formation reveals regions in the nucleotide binding/hinge domain that affect Ca2+ affinity, while mutation of a group of acidic residues in stalk sector 2 also results in lowered Ca2+ affinity. The hypothesis that this acidic stalk sequence interacts with sequences in other domains to control Ca2+ affinity will be tested.